Many forms of heart disease are characterized by a loss of myocytes either by apoptosis or infarction. Therefore, strategies such as cardiac cell transplantation may provide remarkable advances in therapy for a range of heart diseases. A variety of donor cells have been tested in animal models of infarction with the most promising results from adult stem cells. However, embryonic stem (ES) cells may provide the greatest opportunity for myocardial regeneration. The overall goal of this proposal is to utilize nonhuman primate embryonic stem (ES) cells to regenerate infarcted myocardium as an aggressive step toward clinical applications of cardiac cell transplantation. A multidisciplinary approach will provide innovative approaches to address major roadblocks in bringing cell transplantation to the clinic. First, we hypothesize that, with appropriate priming, rhesus ES cells will be able to engraft into the infarcted rhesus myocardium and regenerate multiple cell types typical of myocardium. Specific Aim 1 will test engraftment of donor ES cells labeled with the fluorescent protein EGFP in a rhesus myocardial infarction-reperfusion model. Ex vivo immunohistochemistry will provide information on the cell types regenerated from the donor cells, and isolated myocyte studies will characterize the cellular function of regenerated myocytes. Specific Aim 2 will develop novel imaging applications using Magnetic Resonance Imaging (MRI) to track donor ES cells labeled with magnetodendrimers as well as provide structural and functional details of myocardial regeneration. Next, we hypothesize that the microenvironment of the infarcted myocardium has a rich collection of signaling molecules directing progenitor cell recruitment and milieu-dependent differentiation. In Specific Aim 3, we will search for these signaling molecules in the venous effluent following infarction using antibody-based assays as well as utilizing proteomics technology with mass spectrometry. To test putative signaling molecules in a controlled microenvironment, we will develop a microfluidics bioassay system using cultured ES cells in microchannels. Overall, these developmental approaches have the potential to dramatically advance the field of myocardial regeneration and bring cardiac cell transplantation much closer to clinical reality.